Down-regulation of IL-1beta-induced COX-2 Expression in A549 Lung Cancer Cells at Transcriptional Level by Leptomycin B Involves Inhibition of the IkappaB-alpha/NF-kappaB Pathway but Independent of CRM1

ABSTRACT

PURPOSE: Overexpression of COX-2, an enzyme responsible fro the synthesis of prostaglandins, is well linked to human chronic lung diseases. The mechanism by which COX-2 expression is increased or enhanced in cancer cells remains largely unknown. Any compound which can reduce COX-2 expression may be considered as an anti-cancer agent. MATERIALS AND METHODS: Leptomycin B (LMB) is a metabolite of Streptomyces and a specific inhibitor of CRM1 nuclear export receptor. A549 is a human lung cancer cell line. To evaluate the effect of LMB on COX-2 expression induced by IL-1beta, a pro-inflammatory cytokine, in A549 cells, Western blot and RT-PCR assays were applied to measure COX-2 protein and mRNA expressions in response to IL-1beta, respectively. Luciferase experiments were done to measure promoter activity of COX-2, NF-kappaB or AP-1. CRM1 siRNA trasfection experiment was performed to knock-down endogenous CRM1. Biochemical protein fractionation method was also carried out to see intracellular localization of proteins. RESULTS: LMB at 9 nM strongly suppressed IL-1beta-induced expression of COX-2 protein that was attributable to decreased COX-2 transcript and promoter activity, but not mRNA stability. Distinctly, knock-down of CRM1 had no effect on COX-2 expression by IL-1beta. Moreover, LMB did not affect IL-1beta-induced phosphorylation of ERK-1/2, JNK- 1/2, and p38 MAPK or AP-1 promoter activity. In contrast, LMB blocked IL-1beta- mediated cytosolic IkappaB-alpha degradation, p65 NF-kappaB nuclear translocation, and NF-kappaB promoter activity. CONCLUSION: LMB potently down-regulates IL-1beta- induced COX-2 at transcriptional level in A549 cells, in part, through modulation of the IkappaB-alpha/NF-kappaB pathway but independent of CRM1, MAPKs and AP-1.